Dengue: The Breakbone Fever - Walter Reed Army Institute of ...

Dengue: 

The Breakbone Fever 

WRAIR Tropical 

Medicine Course 

Arthur Lyons, COL, MC 

Viral Disease Branch 

Walter Reed Army Institute of Research

NOT the Band

Disclaimer 

• The points made in this presentation are 

solely the views/opinions of the author and 

do not reflect the views/opinions of the US 

Government, US Department of Defense, 

US Army or the US Army Medical 

Research and Materiel Command.

Take Home Points 

• Mosquito-borne illness 

– Not spread person-to-person 

• First infection can be a bad experience 

• Second infection can be deadly 

• No antiviral treatment 

• No vaccine (yet)

Case (1) 

• 25 y/o male Indiana Jones type presents to your clinic in June @ Ft. Bragg, NC with 

c/o headache, abdominal pain, nausea and vomiting for the past 24 hours. Took 

pepto bismol and tylenol without relief. 

• He returned from leave OCONUS 2 days ago (SubSaharan Africa , Latin America 

and SE Asia) where he swam in the ocean, ate adventurously, suffered numerous 

different insect bites, partook of some “horizontal refreshment” with local talent, and 

volunteered to be a cow herder for 2 weeks in the Pampas. He has 2 cats, a dog, 

tropical fish and several ferrets as pets at home. 3 weeks ago he cleaned out his 

aquarium, and stated it was a “bloody chore”. He did not take appropriate 

prophylaxis prior to/during the trip. 

• What do you do? 

• A) Bellyache, GOMER, discharge 

• B) Give him extra-strength PB and discharge with instruction to f/u with primary HCP 

next week, 

• C) Admit, evaluate for, among other things, malaria, dengue and RMSF 

• D) Consult psychiatry

Introduction to Dengue 

• Definition: Mosquito-borne flaviviral 

disease. 

• Etiology: 

Infection with one of four types of 

dengue virus: 

– DEN-1 

– DEN-2 

– DEN-3 

– DEN-4 

• Transmission: 

– Vector: Aedes mosquito 

• Aedes aegypti 

• Aedes albopictus 

– Blood transfusion 

– Organ transplantation 

– No person-to-person transmission 

documented

History of Dengue 

• Clinical descriptions date as far back as 992 

AD in China 

• David Bylon (Batvia) in 1779 

– “knokkelkoorts” – joint fever 

• Benjamin Rush 

– Termed “breakbone fever” 

– Comes from Swahili “ka dinga pepo” 

meaning a sudden cramp like seizure 

and plague 

Thomas S, Advances in Virus Research 2003. 

Kyle J Annu Rev Microbiol 2008. 62:71-92. 

http://www91.homepage.villanova.edu/eli.greenbaum/peru.htm

History of Dengue (2) 

• Viral etiology suggested in early 1900’s by 

Ashburn and Craig 

• Virus types 1 and 2 isolated during World 

War II 

• 1956 outbreak in Manila led to identification 

of Den-3 and DEN-4 

• Dengue hemorrhagic fever recognized since 

1950’s 

Thomas S Advances in Virus Research 2003. 

Kyle J Annu Rev Microbiol 2008. 62:71-92.

Dengue Virus 

• Flavivirus (single-stranded RNA virus) 

• Spherical, 40-50 nm (dia.) viral particle 

– 3 Structural (E, C, M) proteins 

– 7 Nonstructural (NS1, NS2A, NS2B, NS3, 

NS4A, NS4B, NS5) 

• 4 serotypes 

– DENV 1 through 4 

– Multiple genotypes per serotype 

• Common progenitor 1,000 years ago 

• Serotypes have further divergence 

– 62 to 67% homology based on amino acid sequence 

• Varying pathogenicity based on serotype

Case 2 

• 50 y/o man with multiple mosquito bites 

after exploring the Amazon during a recent 

(2 weeks ago) trip. Had been recently 

web surfing and found out about dengue. 

He asks you if he should take prophylaxis 

against dengue. He has been 

asymptomatic. What do you do? 

• A) Admit, put on ribavirin 

• B) Reassure

Vector 

• Aedes aegypti and Ae. albopticus 

– Highly susceptible to dengue 

• Efficient vectors 

– Prefers human blood 

– Daytime feeder: interrupted, between egg 

laying 

• 0800-1300; 1500-1700 

– Bite goes unnoticed 

– Multiple bites per blood meal; one 

mosquito can infect several persons 

– Adapted to urban life; breeds in freshwater 

containers 

• RAPID TRANSMISSION, EXPLOSIVE EPIDEMICS 

• Rapid 

Aedes 

aegypti

Larvae 

A water sample is teeming with mosquito larvae 

after it was collected from a fountain outside a 

vacant house July 15 in Miami Beach, Fla. 

Miami-Dade County health officials are reporting 

the first suspected local case of dengue fever, a 

potentially serious mosquito-borne illness that 

had once disappeared from the United States. 

http://www.usatoday.com/news/health/2010-08-03-dengue-fever_N.htm

http://www.centromedico.com.uy/Novedades/dengue.htm 

Life Cycle

Habitat 

• Breeds in clean, still, 

stagnant water 

– Discarded tires 

– Water tanks 

– Storage appliances 

http://www.fcen.uba.ar/habitat/dengue.htm

Breeding sites

Dengue: Epidemiology 

Assessment 

• Leading arboviral (mosquito-borne) infection 

• Major health problem in the subtropics and tropics (~35 o N and ~35 o S) 

– Southeast Asia, India, Middle East, Caribbean, Central and South 

America, Australia, South and Central Pacific 

– Transmission in ~ 100 countries 

– Recent suspected dengue outbreaks in 

Yemen, Pakistan, Saudi Arabia, 

Madagascar, 

Sudan, Cape Verde

Epidemiology Assessment (2) 

• 2.5 billion people at risk for infection 

• 50-100 million infections annually 

• ~500,000 cases of DHF annually 

• Up to 25,000 deaths annually 

• Significant Economic Burden 

– SE Asia: 1,300 disability-adjusted life years 

– Similar to TB, other childhood and tropical diseases

World distribution of dengue viruses and their 

mosquito vector, Aedes aegypti, in 2005 

The tropical zone of the world between 350N and 350S latitude and area not over 1,000 ft. 

above sea level is the usual habitat, the areas are marked by monsoon-rains.

http://www.cdc.gov.

Total Dengue Cases and Deaths, 2003-2008 

Source: DengueNet

The Global Resurgence of 

Dengue 

• Unprecedented global population growth 

• Unplanned and uncontrolled urbanization 

• Lack of effective mosquito vector control 

• Globalization of trade 

• More man-made breeding grounds (waste) 

• Increased international air travel 

• Decay in public health infrastructure 

Aedes aegypti in the Americas (1970), at the end of 

the mosquito eradication program, & in 2002

Air Traffic Global Flight Patterns

Departamento de Salud Secretaría Auxiliar de Salud Ambiental 

Programa de Higienización del Ambiente Físico Inmediato 

Directora: Dra. Cano 

Casos Sospechosos de Dengue Semanal, Año 2010 

1 97 97 27 516 5,808 

2 172 269 28 495 6,303 

3 151 420 29 789 7,092 

4 135 555 30 734 7,826 

5 168 723 31 662 8,488 

6 193 916 32 887 9,375 

7 192 1,108 33 1,053 10,428 

8 180 1,288 34 928 11,356 

9 244 1,532 35 

10 193 1,725 36 

11 178 1,903 37 

12 173 2,076 38 

13 192 2,268 39 

14 198 2,466 40 

15 218 2,684 41 

16 151 2,835 42 

17 156 2,991 43 

18 172 3,163 44 

19 156 3,319 45 

20 176 3,495 46 

21 192 3,687 47 

22 235 3,922 48 

23 242 4,164 49 

24 330 4,494 50 

25 384 4,878 51 

26 414 5,292 52

Dengue in the USA 

• Some historical dengue outbreaks in the USA 

– 1780: Philadelphia, PA 

– 1826-8: Savannah, GA 

– 1850-1: Charleston, SC, Savannah, GA, New 

Orleans, LA, Mobile, AL, Galveston, TX, Augusta, GA 

– 1922: Texas, Savannah, GA 

– 1934: Florida 

– 1945: New Orleans 

http://www.topnews.in/number-dengue-cases-delhi-reaches-913-2238269

Dengue in the USA (2) 

• Recent indigenous transmission 

– Texas: 

• 1980: 23 cases, first locally acquired since 1945 

• 1980-1999: 64 cases (lab-documented) 

• 2005: DEN-2 epidemic in Brownsville; estimated incidence of 

recent dengue infection (4% of population) 

– Hawaii: 

• 2001-2002: 122 cases (first since 1944) 

– Florida (Key West): 

• 2009-2011: 93 cases (as of 17 May 2011) 

• 6 cases to date in 2011: Miami-Dade (2), Palm beach (2), 

Martin (1), Hillsborough (1) 1 Counties 

1 

Anil L, Stanek D, Blackmore C, Stark L, Mock V. 

http://www.doh.state.fl.us/Environment/medicine/arboviral/pdfs/2011/2011Week42ArbovirusReport_10-22-2011.pdf

It is here! 

Dengue fever outbreak feared in Key West [Updated] 

July 14, 2010|By Thomas H. Maugh II, Los Angeles Times 

Federal officials said Tuesday that they fear an outbreak of dengue fever in Florida after a 

survey of Key West residents found that at least 5% had been infected or exposed to the 

virus. With the exception of a handful of isolated cases along the Texas-Mexico border, 

there had previously been no cases in the continental United States since 1946 and no 

outbreak in Florida since 1934. 

5% of Key West Population Infected in 2009; 

New Case Suggests Ongoing Outbreak 

By Daniel J. DeNoon 

WebMD Health News 

Reviewed by Laura J. Martin, MD 

May 20, 2010 - An "extended outbreak" of dengue fever is ongoing in Key West, Fla., where some 5% of residents were infected last fall. 

The latest case of the mosquito-borne disease was in mid-April. It's not yet clear whether the April case is a continuation of the 2009 outbreak or a new outbreak from a different dengue strain. 

Although only 28 cases have been definitively identified, blood tests conducted in September 2009 detected evidence of recent infection in 5.4% of 240 randomly selected residents. 

"The best estimate from the survey is that about 5% of the population of Key West was infected in 2009 with dengue," dengue expert Christopher J. Gregory, MD, of the CDC's Epidemic Intelligence Service, tells WebMD.

Key West Dengue 

• RT-PCR done on 1,178 pools of Ae. aegypti 

mosquitoes collected from Monroe County, FL 

from 27 January-17 December 2010 

• DENV-1 

• KW sequence grouped as a member of a large 

clade of recent DV from Central America 

– Nicaragua, 2006, 2008 

• Unknown time of introduction into FL 

Graham AS, Pruszynski CA, Hribar LJ et al., 2011. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 17, No. 11, November 2011

Dengue Impact on U.S. Military 

Operations 

• Philippines 

• World War II 

• Vietnam 

Hospitalized US military personnel, Philippines

Dengue Impact on recent 

U.S. Military Operations 

• Somalia 

– Operation Restore Hope (1993) 

– 58/289 (20%) hospitalized febrile troops had 

laboratory-confirmed DF 

– 69/289 (24%) suspected DF cases 

• Haiti 

– Operation Uphold Democracy (1994) 

– 31/103 (30%) hospitalized febrile troops had DF 

• Defense Medical Surveillance System (DMSS) 

– 1997-2006: 26 DF cases hospitalized, 170 

ambulatory

USASOC Study 

• Seroprevalence study 

• USASOC personnel deployed to dengueendemic 

areas in Latin America 

– At least 30 days, from 2006-2008 

– 500 specimens 

– DoD Serum Repository 

– Sandwich ELISA 

• 11.0% seroprevalence rate 

Caci JB, Lyons AG, Tack DM; ASTMH Abstract # 90, 2010

Pathogenesis 

• Multiple theories regarding pathogenesis but none 

accepted 

• Lack of a reliable animal model 

• Complicated host and viral interactions 

– Different responses in adults and infants 

• Antibody dependent viral enhancement 

– Upregulation of infection 

– Increased cytokine activity 

• Unknown etiology of capillary leak syndrome 

characterized by DSS

Pathogenesis (2) 

• No evidence of direct viral infection of 

endothelial cells 1 

• Transient disruption in the function of the 

endothelial glycocalyx layer 

– A molecular sieve 

– Hypoalbuminemia, proteinuria 

– DENV and NS1 adhere to heparan sulfate 

– Increased urinary heparan sulfate excretion 

seen in kids with severe dengue 2 

1 

Leong AS et al. Semin Diagn Pathol 2007;24:227-36 

2 

Avirutnan P et al. PLoS Pathog 2007;3(11):e183.

Clinical Manifestations 

Dengue Shock 

Syndrome 


Hemorrhagic 

Fever 

50 to 90% of cases 

DEN-2 and DEN-4 

Kyle J Annu Rev Microbiol 2008. 62:71-92.

Clinical Dengue 

– Spectrum of clinical illness 

• Primarily defined in Thai cohorts 

• Asymptomatic infection 50% 

• Undifferentiated fever 

• Dengue fever 

45% 

• Dengue hemorrhagic fever (DHF)/ Dengue 

shock syndrome (DSS) 

Old Classification of Dengue 

http://lukemaciel.deviantart.com/art/Mosquito-da-Dengue-87552543

Old Definition of Dengue 

Hemorrhagic Fever 

• Fever lasting 2-7 days 

• Tendency to hemorrhage 

– Positive tourniquet test (TT) 

– Spontaneous bleeding 

• Platelet count

New Classification 

• Dengue Fever (DF) 

– Classical DF, recovers without major 

sequelae 

• Severe Dengue 

– Plasma leakage resulting in shock 

– Accumulation of serosal fluid 

– Severe bleeding 

– Severe organ impairment 

Dengue: Guidelines for Treatment, Prevention and Control. Geneva, WHO, 2009

Most Dengue infections 

are… 

• Asymptomatic, or 

• Mild symptomatic illness 

–Undifferentiated fever 

–+/- Rash

Dengue Fever (DF) 

• Incubation period 3-7 days, illness 

lasts ~7 days 

• A range of clinical manifestations 

•Three phases 

•Febrile 

•Critical 

•Recovery 

• Epi: Travel/residence in 

urban areas of tropics/sub-tropics 

http://professoralilianbiologia.blogspot.com/2008/04/lilian-cia.html

DF (Day 1, Febrile Phase) 

• Abrupt onset high fever (≥38.5 o C) 

– 5-7 days fever (biphasic) 

• Rash 

– Early flushlike rash may be replaced by a 

macular/morbilliform rash. Late petechiae 

• Chills, vomiting 

• Arthralgias, myalgias 

• Severe headache 

• Eye, Retro-orbital pain 

• Lumbosacral pain 

http://www.cartunista.com.br/romario_dengue.html

DF (Day 2, Febrile Phase) 

– Severe muscle, joint pain 

– Nausea, vomiting 

– Lassitude, prostration 

– Respiratory symptoms 

– Epistaxis, gum bleeding, petechiae 

• Classic DF with some hemorrhage is NOT DHF 

– PE: 

• Fever 

• Generalized rash (may be replaced by macular/morbilliform later 

on). Petechial late 

• Relative bradycardia 

• Generalized lymphadenopathy 

• Petechial hemorrhages 

– After 3-7 days, no sequelae

Rash 

http://www.itg.be/itg/DistanceLearning/LectureNotesVandenEndenE/imagehtml/ppages/CD_1038_061c.htm. Used with permission

Case 3 

• 27 y/o AD USMC from Puerto Rico 

presented with 2 days of increasing fever 

(>38.5 C), severe headache, rash, 

arthralgias, myalgias, while on deployment 

in the Philippines. After 5 days of illness, 

his fever suddenly resolved. Should you: 

• 1) Discharge 

• 2) Draw labs and observe

Symptomatic Dengue Clinical 

Syndromes 

Dengue Fever 

(DF) 


Hemorrhagic 

Fever (DHF) 

Dengue Shock 

Syndrome 

(DSS)

Critical Phase 

• Small proportion of patients 

– Children, young adults 

• Occurs at time of defervescence 

– Around days 4-7 of illness 

• Systemic vascular leak syndrome 

– Increasing hematocrit 

– Hypoproteinemia 

– Pleural effusions 

– Ascites

Severe Dengue 

• Severe plasma leakage 

– Shock (DSS) 

– Serosal fluid accumulation with respiratory distress 

• Severe bleeding 

– Clinically evident 

• Multi-organ involvement 

– Liver: AST/ALT >1000 

– CNS: Impaired consciousness, seizures, 

encephalopathy 

– CV and other

http://www.pattayagogos.com/news08a.htm 

http://denguehemorrhagicfever.tumblr.com/ 

DHF

Dengue Hemorrhagic Fever 

(DHF) 

• Onset as per classical dengue 

• Damage to blood and lymph vessels 

• Defervescence followed (2-5 days) by 

– Ascites, abdominal pain 

– Pleural effusion 

– Hemorrhagic manifestations (gum bleeding, phlebotomy bleeding) 

which may progress to shock 

– Central cyanosis 

– Diaphoresis 

• Epi: Exposure in dengue endemic region with possible previous 

dengue infection

• PE: 

– Restlessness 

– Abdominal pain 

– Hemorrhage 

• Petechiae 

• Spontaneous ecchymoses 

DHF (2) 

• Bleeding: GI, GU, phlebotomy 

– Tender hepatomegally (75%), splenomegally 

– Pleural* effusions (80%) perirenal effusions (77%), hepatic, 

splenic, pericardial, peritoneal effusions* (variable%) 

– Shock 

• Rapid, weak pulse 

• Pulse pressure

Lab 

• Positive tourniquet test (or hemorrhagic manifestations) 

• Thrombocytopenia (20%)* 

– Pleural effusion/ascites 

– Petechiae 

• Hepatorenal shutdown with shock 

• Viral isolation from acute serum 

• Convalescent IgM (+) 

• Peak proteinuria** 

– 0.56 v. 0.08 g/d (P

Dengue Shock Syndrome 

(DSS) 

• Fluid leak outside of blood vessels 

• Lasts 1-2 days 

• Massive hemorrhage 

• Shock, peripheral vascular collapse 

– Hypoperfusion c/b myocardial dysfunction: metabolic acidosis 

and MOF 

• Cyanosis, massive pleural effusions, ascites 

• Narrowing pulse pressures (

Subcutaneous 

hemorrhage in DHF 

http://www.orble.com/aia/

Risk Factors for DHF/DSS 

• Pre-existing immunity from previous infection (heterogenous subtype) 

• Diabetics, asthmatics, other chronic diseases 

• DENV type 

– DENV-1,3 > 2,4 

• Increased time between infections 

• Under age 15 

– Increased capillary fragility 

• HLA type and race* 

– Caucasian>AA 

– HLA Class-1 alleles 

• Female sex 

• AB blood group 

• Promotor variant of DC-SIGN receptor 

• Single-nucleotide polymorphism in TNF gene 

*De la C Sierra B, Kouri G, Guzman MG. Arch. Virol., 2007, 152(3) 533-42. Epub 2006 Nov. 16.

Factors that reduce the risk of 

severe dengue 

• Race 

• Second or third degree malnutrition 

• Polymorphisms in the Fc-gamma and 

Vitamin D receptor genes

Criteria For Dengue +/- 

• Probable case: 

Warning Signs 

– Resident/travel to dengue endemic area and 

2 of the following: 

– Nausea, vomiting 

– Rash 

– Aches and pains 

– + TT 

– Leukopenia 

– Any warning sign

Warning Signs 

• Continual/increasing abdominal pain/tenderness 

• Persistent vomiting 

• Clinical fluid accumulation (serosal) 

• Mucosal bleeding 

• Lethargy, restlessness 

• Tender hepatomegaly (>2 cm), ascites 

• Lab: increase in Hct. concurrent with rapid 

decrease in platelets* 

• Sudden reduction in temperature

Recovery Phase 

• Altered vascular permeability syndrome 

resolves 

– After 48-72 hours 

• Rapid improvement in patient’s symptoms 

• Rash 

– Mild maculopapular to severe, pruritic lesions 

(leukocytoclastic vasculitis) 

– Resolves with desquamation (1-2 weeks) 

• Profound fatigue for several weeks

Case 4 

• 30 y/o AD Sailor who recently returned 

from a TDY to Thailand 4 days ago. Has 

had 2 days of fever, excruciating HA, eye 

pain, severe myalgias, arthralgias, sweats 

and rash. You suspect dengue. 

• How to diagnose? 

• How to treat?

Lab 

• Marked leukopenia 

• Thrombocytopenia 

• Moderate elevation of AST/ALT 

• Viral isolation to Day 5 only 

• Negative malaria smears 

• Dengue IgM (+) on Day 6 serum 

– Takes 5 days to manifest 

• PCR available 

• Convalescent: 4-fold rise in IgG may be required

Lab (2) 

• Antibody specificity increases over time 

• Most readily available diagnostic tests 

– ELISA (serology) 

• Cross-reactive; not specific 

• MAC-ELISA, IgG ELISA 

• IgM/IgG (>1.2, 1.4?) not defined 

– PRNT, microneutralization (serology) 

• More specificity 

• Research, vaccine work 

– Viral Isolation 

• Most specific

Lab (3) 

• Nucleic Acid Amplification (NAAT) 

– RT-PCR 

– Real Time RT-PCR 

– NASBA 

– None commercialized to date 

– None standardized 

• Antigen Detection 

– NS1 

– Antigen capture ELISA, lateral flow antigen detection, NS1 IgM, 

IgG responses. 

– Do not differentiate between the different serotypes

Slide courtesy of Dr. Subhamoy Pal 

Immune Response to Dengue 

NS-1: Effective days 1-5 

post onset of symptoms 

Acute 

Acute 

IgM/IgG: Effective after 

day 5 

Convalescent 

Convalescent 

A diagnostic capable of 

detecting both is 

desirable 

Day 0 7

Lab (4) 

• Primary infection 

– IgM first to appear, at end of 3-5 day fever period (~50%), day 6- 

10 (93-99%), peak (2 weeks), undetectable by 2-3 mos. 

– IgG appears at end of first week of illness, persists for >year 

– RT-PCR can provide a same-day diagnosis with a similar 

sensitivity to culture 

• Secondary infection 

– IgM typically LOWER titer than primary infection; false negatives 

have occurred 

– IgG typically HIGHER titer than primary infection; may x-react 

with other flaviviruses (JE, YF, WN)

Criteria for Confirmed and 

Probable Dengue Infection 

• Confirmed 

– Viral isolation 

– Genome detection 

– Antigen detection 

– IgM/IgG seroconversion 

• Probable 

– IgM positive 

– Elevated IgG titer (> 1,280 by HAI)

http://www.cdc.gov/dengue/clinicalLab/diagnosticProcess.html 

Diagnosis

Tests Used for the Lab Diagnosis of 

Primary Dengue Infection 

Test 

Diagnostic 

Window 

Sample 

Required 

Sample 

Storage 

Turnaround 

Time 

Viremia 

(Culture) 

Acute Phase 1 mL -80°C 2 weeks 

RT-PCR Acute Phase 140 μL Refrigerate if 

, - 

20°C 

1-2 days 

IgM ELISA 

Day 4 to –Day 

90 post 

infection 

1 mL Frozen or 

refrigerated 

1-2 days 

IgG ELISA Day 14 to > 1 

year postinfection 


refrigerated 

1-2 days 

PanBio 

duoCassette 

Day 4 to Day 10 μL Refrigerate if 

, - 

20°C 

1-2 days 

Serum 

Neutralization 

(PRNT) 

1 week to >1 

year postinfection 


refrigerated 

1 week 

SST or red top tube 

Virus isolation in cell culture and detection by IFA 

Used with IgG ELISA to differentiate primary from secondary infection 

Used with IgM ELISA to differentiate primary from secondary infection

Rapid Diagnostic Tests 

(RDT’s) 

Current RDT’s 

Future RDT’s 

Important for: 

• Quick diagnosis (lab results take time 

and require labs) 

• In resource-limited settings 

• Alerts a unit to ID threats 

• Helpful for triage during outbreaks 

• Curtail geographic spread of 

infectious diseases 

• Stability operations and 

infrastructure building 

Worldwide demand for better diagnostics to 

manage treatment and prevention 

Slide courtesy of Dr. Subhamoy Pal

Current Rapid Diagnostic 

Technologies 

Agglutination 

Flow through 

Lateral Flow 


Solid Phase 

Isothermal Nucleic Acid Tests

Product Introduction 

#1: IgG/IgM Dengue Duo Cassette 

10μL of serum, plasma, or whole 

blood 

15 minutes (time to result) 

Wu et. al. CDLI 2000, pp 106-109 

10 uL of serum 

1.5 hours 


Product Introduction cont'd 

#2: NS-1/IgG/IgM Dengue Duo 

Cassette 

120μL of serum or plasma 

15 minutes (time to result) 

Osorio et al. Virology Journal 2010, 7:361 


Ag/Ab level 

Ag/Ab level 

Standard Diagnostics Dengue Duo (NS-1) RDT 

NS1 Ag 

3 drops (110 μl) of plasma or serum 

for early acute phase samples (day 1 ~5) 

IgG/IgM Ab 

10 μl of plasma or serum for early convalescence 

phase samples (after day 5 ~ 14) 

IgG 

IgG 

NS1 Ag 

IgM 

NS1 Ag 

IgM 

1 2 3 5 7 10 12 Day 1 2 3 5 7 10 12 

Day 


Interpretation 

Negative 


Primary 

Secondary

Why make a primary/secondary 

determination? 

• The majority (>90%) of DHF/DSS cases are secondary infections 

– One 20 year longitudinal study suggests that among all DHF/DSS 

cases 9% are primary and 91% are secondary (Nisalak, A., et al., 

Am J Trop Med Hyg, 2003. 68(2): p. 191-202) 

• Overall, 2-4% of secondary infections proceed to severe dengue. 

Other risk factors also need to be considered (Guzman, M.G., et 

al.,1997. Am J Epidemiol, 2000. 152(9): p. 793-9; discussion 804) 

Positive predictive value of 

secondary infection leading to 

DHF varies by region and 

attack rate. 


Commercially Available NS-1 

• Rapid Tests 

Products 

– Bio-Rad StripTM 

– SD (Focus) BIOLINE Dengue NS1 Assay 

– SD (Focus) BIOLINE Dengue Duo IgM/IgG/NS1 

Assay 

– Panbio Dengue Early Rapid 

• ELISA format 

– Panbio Dengue Early PanE (2 nd Generation) 

– SD NS-1 Dengue Ag ELISA 

– Bio-Rad PlateliaTM Dengue NS1 Ag 

(Evaluated by NMRC) 


Assay specifications 

Parameters SD Rapid Biorad 

Rapid 

Panbio 

Rapid 

BioRad 

ELISA 

Panbio 

ELISA 

Number of 1 2 3 5 7 

steps 

Blood EDTA-treated Plasma, Sera Plasma, Sera 

matrices blood, 

plasma, sera 

sera 

sera 

Assay Time 15-20 

minutes 

15-30 

minutes 

15 minutes 140 

minutes 

160 

minutes 

Volume 105 uL 50 uL 50 uL 50 uL 75 uL 

necessary 

Format Cassette Dipstick Dipstick 96-well 96-well 

Extra 

materials 

required 

No 

Tubes, 

pipette 

Pipette 

Pipette, 

incubator 

, plate 

reader 

Pipette, 

incubator 

, plate 

reader 

Storage Room Temp. 2-8°C 2-8°C 2-8°C 2-8°C 


Summary of data 

Panbio Dengue IgM/IgG Duo Cassette RDT 

- Down-selected from among several Dengue RDT’s 

- Meets KSA and Attributes of draft CDD 

- Marketed overseas with record of stability 

- Ideal after day 5 post-onset of symptoms 

SD NS-1 Cassette RDT’s developed recently 

- Available NS-1 RDT’s comprehensively evaluated 

- Meets KSA and Attributes of draft CDD 

- Required for early diagnosis of dengue between day 0-7 

post-onset of symptoms 

Together, the two RDT’s can enable dengue diagnosis through all 

stages of infection to fulfill capability gap. 


Advantages and limitations 

of different dengue diagnostic tests 

Diagnostic tests Advantages limitations 

Viral isolation and identification 

RNA detection 

• Confirmed infection 

• Specific 

• Identifies serotypes 


• Sensitive and specific 

• Identifies serotype and genotype 

• Results in 24–48 hours 

• Requires acute sample (0–5 

days post onset) 

• Requires expertise and 

appropriate facilities 

• Takes more than 1 week 

• Does not differentiate between 

primary and 

secondary infection 

• Expensive 

• Potential false-positives owing to 

contamination 

• Requires acute sample (0–5 

days post onset) 

• Requires expertise and 

expensive laboratory 

equipment 

• Does not differentiate between 

primary and 

secondary infection

Advantages and limitations of 

different dengue diagnostic tests: Serology 

Diagnostic Tests Advantages Limitations 

IgM or IgG seroconversion 

IgM detection (single sample) 


• Least expensive 

• Easy to perform 

• Identifies probable dengue 

cases 

• Useful for surveillance, 

tracking outbreaks 

and monitoring effectiveness of 

interventions 

• IgM levels can be low in 

secondary infections 

• Confirmation requires two or 

more serum samples 

• Can differentiate between 

primary and secondary 

infection* 

• IgM levels can be low in 

secondary infections 

*Primary infection: IgM-positive and IgG-negative (if samples are taken before day 8–10); secondary infection: IgG should be higher 

than 1,280 haemagglutination inhibition in convalescent serum.

Advantages and limitations of 

different dengue diagnostic tests: Antigen 

Detection 

Diagnostic Test Advantages Limitations 

Clinical specimens (for 

example, using 

blood in an NS1 assay) 

Tissues from fatal cases (for 

immunohistochemistry, 

for example) 


• Easy to perform 

• Less expensive than virus 

isolation or RNA 

detection 


• Not as sensitive as virus 

isolation or RNA detection 

• Not as sensitive as virus 

isolation or RNA detection 

• Requires expertise in 

pathology

Tourniquet Test (TT) 

• Positive in up to 50% of patients with 

classical dengue and almost all with DHF 

• Non-specific 

• Procedure: 

– Inflate BP cuff halfway between systolic and 

diastolic BP for 5 minutes 

– Release 

– Count # petechiae in a quarter-sized patch 

below the cuff 

– >20 is positive

Sample Prep 

• Collect 2 separate red gel separator tubes (“tiger-tops”) 

– Gently invert 5 times 

– Allow blood to clot min. 30 min (vertical) 

– Centrifuge at full speed (1100-1399 G) for 10 min 

– Pipette off serum into separate cryovials 

– Refrigerate or ice bath (2-8°C, ELISA/PRNT) 

– RT-PCR: store @ 2-8°C for up to 6 hours (immediate RNA 

extraction possible) otherwise, store @ -20°C for up to 14 days. 

Limit to one freeze-thaw cycle. 

– Isolation: store @ -80°C until ready for transport

Rx 

• Symptomatic, supportive 

– CAREFUL fluid management 

• Acetaminophen 

– NO Aspirin (Reye’s) 

• Oral Fluid Replacement 

– If can take PO, no complications, mentally with it 

• Serial monitoring of (to trigger IV therapy) 

– HR, BP, Skin perfusion, Urine output, Hct (>20%) 

• Development of any warning sign 

– Hospitalization, close observation 

– Judicious use of IV fluids on poor PO, rapidly increasing Hct.

Rx (2) 

• Shock 

– PROMPT fluid resusctiation 

– Isotonic crystalloid (initially)/colloid (for those presenting in profound 

shock, no response to crystalloid) solutions 

– Keep to minimum required to support CV stability 

– Plasma, cryoprecipitates, whole blood (care to not fluid overload) 

– PREVENTIVE transfusions should be avoided 

• Desmopressin? IV gamma globulin? Steroids? Drugs (chloroquine, 

balapiravir, statins)? No evidence for efficacy 

• Beware pulmonary edema: may need PPV 

– DHF-DSS is the 3 rd most common cause of ARDS in hospitalized children in 

Malaysia


Prevention 

• Prevention: 

– There is no prophylactic drug for dengue 

– There is no licensed vaccine (yet) to prevent dengue 

– Reduce risk by use of personal protective measures (DEET, 

permethrin-treated uniforms, screened windows, mosquito 

netting) and local vector control (eliminate breeding sites, 

insecticides) 

– New approaches to vector control 

• Genetically altered male mosquitoes 

• Embryonic introduction of wolbachia into A. aegypti

Prevention 

http://www.rafaela.gov.ar/es/popupDenge2.html 

http://wahootours.hyperboards.com/action/view_topic/topic_id/5675

And now for something 

completely different… 

• 31 y/o female recently returned from Singapore… 

• Fever (39.5°C), nausea, myalgias, back pain, HA, 

bilateral conjunctivitis, severe bilateral arthralgias 

(shoulders, knees, ankles, elbows, wrists, fingers). 

• Lab: Lymphopenia (0.6 G/L), AST 177 UI/L, ALT 116 

UI/L, LDH 780 UI/L, Nl Bili, CRP 64 mg/L. 

• Course: developed chronic distal arthritis and 

tenosynovitis, swelling of the joints without fluid 

accumulation.

Chikungunya

Summary 

• Dengue is a significant threat to the US military 

and civilian populations in endemic areas. 

– Recognize atypical presentations: maintain healthy 

suspicion 

– May not have high case fatality rates, but illness will 

significantly affect mission(s) 

• Vaccine development is underway and is 

challenging 

– WRAIR is a leader in developing dengue vaccines 

– Several candidate vaccines are in the pipeline

“Most individuals in the United States are 

• as Widespread little concerned dengue about a real dengue possibility fever as 

they were a decade ago about West Nile 

states) fever. That situation could change if 

dengue • Reemerged continues South its expansion and Central America, as one of 

the Caribbean, world’s and most Puerto aggressive Rico reemerging 

• Increased outbreaks infections.” 

Texas and Hawaii 

• Increasing presence of Aedes albopictus (36 

• Vaccine is needed but far from being ready 

Morens D and Fauci A. JAMA 2008. 299:214-216.

Viral Disease Branch 

Walter Reed Army Institute 

of Research 

Division of Viral Diseases

Dengue: The Breakbone Fever - Walter Reed Army Institute of ...

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